Amphoteric sterols and the use thereof

ABSTRACT

An amphoteric compound based on a sterol skeleton, the 3-position of the sterol ring system being substituted by one or more amphoteric groups having an isoelectric point of between about 4 and 9, together with liposomes containing such compounds and their uses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage of PCT/EP02/01878 filed Feb. 21,2002. Priority is claimed from German Patent Application Number 101 09898.7 filed Feb. 21, 2001.

FIELD OF THE INVENTION

The invention relates to amphoteric compounds based on a sterolskeleton, the 3-position of the ring system being substituted by one ormore amphoteric groups with an isoelectric point of between 4 and 9, andto liposomes containing such compounds.

BACKGROUND OF THE INVENTION

The term “lipids” summarizes three classes of natural materials whichcan be isolated from biological membranes: phospholipids, sphingolipids,and cholesterol, including its derivatives.

These substances are of technical interest in the production ofliposomes. Inter alia, such liposomes can be used as containers foractive substances in pharmaceutical preparations. In such uses,efficient and stable packaging of the cargo and controllable release ofthe contents are desirable. Both of these requirements are not easy tocombine: the more stable and compact the packaging, the more difficultthe release of the entrapped active substance therefrom. For thisreason, liposomes changing their properties in response to an externalstimulus have been developed. Thermosensitive and pH-sensitive liposomesare well-known. The pH-sensitive liposomes are of special interest,because this parameter undergoes changes even under physiologicalconditions, e.g. during endocytotic reception of a liposome in a cell,or during passage of the gastrointestinal tract. According to the priorart, pH-sensitive liposomes particularly comprise cholesterolhemisuccinate (CHEMS).

Cholesterol hemisuccinate, in mixture with phosphatidyl ethanolamine, isused to produce pH-sensitive liposomes (Tachibana et al. (1998); BBRC251, 538-544, U.S. Pat. No. 4,891,208). Such liposomes can enter cellsby endocytosis and are capable of transporting cargo molecules into theinterior of cells on this route, without doing damage to the integrityof the cellular membrane.

One substantial drawback of CHEMS is its anionic character. Liposomesproduced using same have a negative overall charge, being taken up bycells with low efficiency. Despite the transfer mechanism describedabove, they are barely suitable for the transport of macromolecules intocells.

In WO 00/59474, the prior art describes compounds having a membraneanchor and a cationic and anionic head group, the anionic group beinglinked to the basic structure via a disulfide bridge. The disulfidebridge can be reduced under physiological conditions, e.g. by contactwith the cytosol, the anionic head group then is liberated, and theoverall molecule assumes a positive charge, thereby enabling fusion withthe cell membrane. The toxicity profile and storage stability of thecompounds disclosed in WO 00/59474 are disadvantageous, because cleavageof the disulfide bridges results in free cationic lipids.Disadvantageously, these compounds are known to have a cytotoxic effect.

For the transport of active substances into cells (transfection), theart uses cationic liposomes having a preferably high and constantsurface charge. The positive overall charge of such particles leads toelectrostatic adherence to cells and subsequently to efficient transportinto same. The use of these compounds and of liposomes produced usingsame remains restricted to in vitro or ex vivo applications, becausesuch positively charged liposomes result in uncontrolled formation ofaggregates with serum components.

SUMMARY OF THE INVENTION

The object was therefore to produce new compounds,

-   i) by means of which active substances can be entrapped in liposomes    and released therefrom when changing the pH value;-   ii) the presence of which aids to achieve the production of    amphoteric liposomes which can be mixed with serum under    physiological conditions, with no aggregation taking place; and-   iii) by means of which liposomes capable of transporting an    entrapped active substance into the interior of cells can be    produced.

Another object of the invention was to find a way allowing easy andlow-cost production of said compounds and incorporation thereof in highamounts in liposomal membranes.

The object of the invention is accomplished by means of sterolderivatives with an isoelectric point of between 4.5 and 8.5,represented by the general formulaAmphoteric substance -Y- spacer -X- sterol,wherein Y and X represent linking groups.

The object of the invention is accomplished by conjugating amphotericgroups to the 3-position of a sterol skeleton. Depending on theamphoteric substance used, compounds are obtained which undergo changesin their charge at a specific pH value and allow incorporation thereofin liposomal membranes in surprisingly high amounts. Ordinary andinexpensive sterols or derivatives thereof can be used as startingcompounds.

Situated between the amphoteric substance and the sterol skeleton arethe molecule fragments: -Y-spacer -X. For example, the spacer is a loweralkyl residue of linear structure, which has from 0 to 8 C atoms andincludes 2 ethylenically unsaturated bonds. The spacer may comprisehydroxyl groups to increase the polarity of the molecule.

DETAILED DESCRIPTION

In the context with this invention, the following abbreviations will beused:

CHEMS Cholesterol hemisuccinate PC Phosphatidyl choline PE Phosphatidylethanolamine PS Phosphatidyl serine Hist-Chol Histidylcholesterolhemisuccinate

Among the membrane-forming or membrane-bound groups of a biologicalbilayer membrane, the sterols are of special interest because thesecompounds, in particular, are available at low cost, involve ordinarychemistry, and allow incorporation in membranes in high amounts withoutincreasing the permeability thereof or even completely destroying theirmembrane character. However, in order to retain this latter feature, itis important that substitution with a polar molecule be at the3-position of the sterol.

The overall molecule assumes its pH-dependent charge characteristics bythe simultaneous presence of cationic and anionic groups in the“amphoteric substance” molecule portion. More specifically, anamphoteric substance is characterized by the fact that the sum of itscharge components will be precisely zero at a particular pH value. Thispoint is referred to as isoelectric point. Above the pI the compound hasa negative charge, and below the pI it is to be regarded as a positivecation, the pI of the compounds or sterol derivatives according to theinvention ranging between 4.5 and 8.5.

The overall charge of the molecule at a particular pH value of themedium can be calculated as follows:z=Σni·((qi−1)+(10^((pK−pH))/(1+10^((pK−pH)))))qi: absolute charge of the ionic group below the pK thereof (e.g.carboxyl=0, single-nitrogen base=1)ni: number of such groups in the molecule

For example, a compound according to the invention is formed by couplingthe amino group of histidine to cholesterol hemisuccinate. At a neutralpH value of 7, the product has a negative charge because the carboxylfunction which is present therein is in its fully dissociated form, andthe imidazole function only has low charge. At an acid pH value of about4, the situation is reversed: the carboxyl function now is largelydischarged, while the imidazole group is fully protonated, and theoverall charge of the molecule therefore is positive.

In a preferred embodiment of the invention, the sterol derivative has anisoelectric point of between 5 and 7.

In another preferred embodiment of the invention, the amphotericsubstance has one or more cations with a pKa value of between 4 and 8and, at the same time, one or more anions with a pKa value of between 3and 7. An advantageous selection as to type and number of functionalgroups can be made with reference to the above-mentioned formula.

In particular, it is convenient to use functional groups or moleculefragments as charge carriers, which are in dissociated form in a pHrange between 4 and 9. For example, these include phosphoric acidgroups, sulfonic acid groups or other strong anions. However, these alsoinclude primary, secondary or tertiary amino groups, unless mentionedabove. These include quaternary ammonium, amidinium, pyridinium, andguanidino groups.

These fixed charges then can be overcompensated by the variable chargesdescribed above. That is, the variable charges are used in excess, forexample. One advantage when using fully dissociated groups is theirstrong polarity. Preferred structural fragments from this group aresulfonic acids, phosphoric acids, primary, secondary or tertiary amines,ammonium compounds, guanidinium compounds, amidinium compounds, orpyridinium compounds linked to the molecule by one of theabove-mentioned spacers and coupling groups.

In a particularly preferred fashion, the amphoteric substances can be inthe form of complete structural moieties. For example, this is the casewith o-, m- or p-aminobenzoic acids, imidazolecarboxylic acid,imidazolediacetic acid, but also nicotinic acid or picolinic acid. Inparticular, the amphoteric substances can be composed of two chargecarriers which both change their charge in the above-mentioned pH rangeof between 4 and 9. The simultaneously occurring loss of anionic chargeand gain of cationic charge results in a change of charge in the overallmolecule.

In another preferred embodiment of the invention, the cation comprisesan imidazole, piperazine, morpholine, purine, and/or pyrimidine. Otheradvantageous cations having this property essentially include all theother nitrogen bases. Particularly in those cases where the nitrogenbases are in the form of a ring system, positional isomersadvantageously are existing, wherein the linking spacer is substitutedto various positions of the organic cation. Conveniently, the pKa valuesof the organic cation can be influenced via said positional isomerism.The relevant fundamental rules are well-known to those skilled in theart. Alternatively, these effects can be estimated from tabularcompilations (Handbook of Chemistry and Physics, Vol. 73, pp. 8-37ff.).Coupling advantageously results in amphiphilic organic cations, e.g.those derived from the following classes of substances:

o-, m-, p-anilines; 2-, 3- or 4-substituted anisidines, toluidines orphenetidines; 2-, 3-, 5-, 6-, 7- or 8-substituted benzimidazoles, 2-,3-, 4- or 5-substituted imidazoles, 1- or 5-substituted isoquinolines,2-, 3- or 4-substituted morpholines, 2-, 3- or 4-substituted picolines,1-, 2- or 3-substituted piperazines, 2-, 5- or 6-modified pterines, 3-,4-, 5-, 6- or 9-substituted purines, 2- or 3-substituted pyrazines, 3-or 4-substituted pyridazines, 2-, 3- or 4-modified pyridines, 2-, 4-, 5-or 6-substituted pyrimidines, 1-, 2-, 3-, 4-, 5-, 6- or 8-substitutedquinolines, 2-, 4- or 5-substituted thiazoles, 2-, 4- or 6-substitutedtriazines, or derivatives of tyrosine. Particularly preferred are theabove-mentioned piperazines, imidazoles and morpholines, purines orpyrimidines. Highly preferred are molecule fragments such as occurringin biological systems, i.e., for example: 4-imidazoles (histamine,histidine itself), 2-, 6- or 9-purines (adenine, guanine, adenosine, orguanosine), 1-, 2- or 4-pyrimidines (uracil, thymine, cytosine, uridine,thymidine, cytidine), or pyridine-3-carboxylic acids. Theabove-mentioned structural fragments may also have additionalsubstituents. For example, these can be methyl, ethyl, propyl, orisopropyl residues, more preferably in hydroxylated form, including oneor two hydroxyl groups. Also, these can be hydroxyl or keto functions inthe ring system.

For example, nitrogen bases with preferred pKa values are also formed bysingle or multiple substitution of the nitrogen atom with loweralkanehydroxyls such as hydroxymethyl or hydroxyethyl groups. Suitableorganic bases from this group are e.g. aminopropanediols,triethanolamines, tris(hydroxymethyl)methylamines,bis(hydroxymethyl)methyl-amines, tris(hydroxyethyl)methylamines,bis(hydroxyethyl)-methylamines, or the corresponding substitutedethylamines.

In another advantageous embodiment, the anionic charge carriers arecarboxyl groups. Naturally, any carboxylic acid can be used as chargecarrier. In particular, these include aliphatic straight-chain orbranched carboxylic acids with up to 8 C atoms and 0, 1 or 2ethylenically unsaturated bonds. Exemplary components of compounds arethe carboxyl group itself, acetic acid, bromoacetic acid, chloroaceticacid, acetoacetic acid, propionic acid, acrylic acid, butyric acid,crotonic acid, or higher carboxylic acids bound to the aliphatic chain,dicarboxylic acids such as oxalic acid, malonic acid, succinic acid,maleic acid, fumaric acid, malic acid, tartaric acid, glutaric acid,adipic acid, caprylic acid, pimelic acid, suberic acid,cyclohexanedicarboxylic acid or cyclopentanedicarboxylic acid,mono-esterified or amidated or bound to the aliphatic chain,oligocarboxylic acids such as citric acid, isocitric acid orethylenediaminetetraacetic acid, mono-esterified or amidated or bound tothe aliphatic chain.

Other advantageous components of compounds are glycolic acid, lacticacid, hydroxybutyric acid, malic acid, tartaric acid, aspartic acid, orglutamic acid, alanine, glycine, serine, threonine, asparagine,glutamine, proline, tyrosine, or cysteine, or other amino acids orhydroxy acids bound to the side chain via a heteroatom.

Carboxylic acids with suitable properties can also be found assubstituents of aromatic systems, e.g. as benzoic acid, anisic acid, o-,m- or p-hydroxybenzoic acid, as dihydroxybenzoic acid, gallic acid,cinnamic acid, phenylacetic acid, hippuric acid, phthalic acid,terephthalic acid, 2-, 3- or 4-pyridinecarboxylic acid, furancarboxylicacid. Other anionic groups are dissociable hydroxyls or thiols, such asoccurring in ascorbic acid, N-substituted alloxan, N-substitutedbarbituric acid, in veronal, phenol, or as a thiol group.

In a preferred embodiment of the invention, the amphoteric substancesare peptides including from 1 to 10 amino acids. In another embodiment,particularly the amino acids histidine, arginine, lysine, glutamic acid,or aspartic acid are used in a particularly preferred fashion to formthe amphoteric substance and determine the charge characteristicsthereof. Other preferred amino acids are glycine, serine, threonine,glutamine, asparagine, but also cysteine, which contribute to increasethe polarity and thus enhance the solubility of the amphotericsubstance.

In a preferred embodiment of the invention, the sterols are cholesterol,sitosterol, campesterol, desmosterol, fucosterol, 22-ketosterol,20-hydroxysterol, stigmasterol, 22-hydroxycholesterol,25-hydroxycholesterol, lanosterol, 7-dehydrocholesterol,dihydrocholesterol, 19-hydroxycholesterol, 5α-cholest-7-en-3β-ol,7-hydroxycholesterol, epicholesterol, ergosterol, and/ordehydroergosterol, as well as other related compounds. The sterols usedwith advantage as starting materials may bear various groups in the3-position thereof, which groups conveniently allow for ready and stablecoupling or optionally assume the function of a spacer. Particularlysuitable for direct coupling are the hydroxyl group which is naturallypresent, but also, the chlorine of sterol chlorides, or the amino groupof sterolamines, or the thiol group of thiocholesterol.

In a preferred embodiment of the invention, the linking group Xcomprises the structures —(C═O)—O—; —(C═O)—NH—; —NH—(C═O)—O—; —(C═O)—S—;—O—; —NH—; —S—; or —CH═N—. Advantageously, the linking group Y maycorrespond in its structure to the group X, and may additionallycomprise the structures —O—(O═C)—; —S—(O═C)—; —NH—(O═C)—; —O—(O═C)—NH—;or —N═CH—. For example, the Y group can be omitted in those cases wherethe amphoteric substance can be coupled directly to the sterol skeleton,e.g. in the esterification of imidazole-4,5-dicarboxylic acid withcholesterol.

In a preferred embodiment of the invention, the spacer is a lower alkylresidue of linear, branched or cyclic structure, which has from 0 to 8 Catoms and includes 0, 1 or 2 ethylenically unsaturated bonds. The spacermay have hydroxyl groups so as to increase the polarity of the molecule.In particular, the spacer can be a sugar, and advantageously apolyethylene glycol which may comprise up to 20 monomer units.

Methods of performing such coupling reactions are well-known to thoseskilled in the art and may vary depending on the starting material andcoupling component employed. Typical reactions are esterification,amidation, addition of amines to double bonds, etherification, orreductive amination.

Particularly preferred molecules can be produced by amidation ofcholesterol hemisuccinate, or by formation of a carbamoyl fromcholesterol chloroformate, and also by direct esterification withcholesterol. Particularly preferred amphoteric substances include, forexample, the following compounds in Table 1, wherein R₁ or R₂ representthe lipophilic portion of the amphoteric sterol, and ( )_(n) representsother portions of the molecule in the sense of the above-defined spacer.

TABLE 1

Histidine derivatives.Coupling of the sterol preferablyproceeds via theamino group as R₂.In this event, R₁ is an anion andcan be e.g. H orahydroxycarboxylic acid or one ormore amino acids. Wherecouplingproceeds via R₁, R₂ is an anionicresidue, e.g. a carboxylic acidordicarboxylic acid.

Piperazine derivatives.Coupling of the sterol may proceedvia one of thering atoms. In thosecases where the side chains arehydroxycarboxylicacids or aminoacids, coupling may proceed withadvantage via theseheteroatoms.

Morpholine derivatives.Coupling of the sterol may proceedvia one of thering atoms. In thosecases where the side chains arehydroxycarboxylicacids or aminoacids, coupling may proceed withadvantage via theseheteroatoms.

Derivatives of Imidazole-4,5-di-acetic acid.Coupling of the sterolpreferablyproceeds as an ester via any of thetwo acetic acid functions.Thesterol may also be bound via the3-amino function.

Piperidine derivatives.Coupling of the sterol may proceedvia any of thering atoms. In thosecases where the side chains arehydroxycarboxylicacids or aminoacids, coupling may proceed withadvantage via theirheteroatoms.

Diaminobenzoic acid derivatives.Coupling of the sterolpreferablyproceeds via any of the two aminogroups. The second aminogroup canbe alkylated, for example, so as toobtain a higher pKa value.

Nitrilotriacetic acid derivatives.Amphoteric groups are also formedbyesterification ofnitrilotriacetic acid. In addition,the chargeproperties of suchcompounds can be modified bycomplexing of metal ions.

N-Alkylcarboxyamino acidderivatives.

Amphoteric compounds are alsoformed by coupling of sterols totheterminal groups of N-acylaminoacids. Advantageously, thestructure can bederived fromserine, aspartic acid or glutamicacid, or from lysine orornithine.

Coupling of the aminodicarboxylicacids not only can be at theterminus,but also at the otheracid groups. In addition, thecharge properties ofsuch compoundscan be modified by complexing ofmetal ions.

EDTA derivatives.Amphoteric groups are also formedby esterificationofethylenediaminetetraacetic acid. Inaddition, the charge propertiesofsuch compounds can be modified bycomplexing of metal ions.

The invention also relates to liposomes comprising the sterolderivatives according to the invention. The compounds of the inventioncan be incorporated in high amounts in liposomal membranes, resulting ina positive charge of the overall particle only when the pH value of themedium drops below the isoelectric point of the amphoteric substance.Liposomes comprising the components of the invention can be coated withpolymers under suitable conditions, where single or multiple depositionof such substances on the surface is possible. In multiple deposition,optionally in the presence of crosslinkers, liposomal nanocapsules areformed as described in WO 00/28972 or WO 01/64330. One advantageous factwhen using the substances described herein is that the electrostaticinteraction with the polyelectrolyte can be interrupted. As iswell-known, the interaction of a polyelectrolyte with charge carriers ofthe liposomal membrane may give rise to demixing of membrane componentsand formation of lipid clusters. In many cases, such demixing isaccompanied by a permeabilization of the liposomes. The substances ofthe invention allow for elimination of this interaction following thecoating process. When increasing the pH value at this point, theliposomes will be entrapped in the nanocapsules merely in a stericfashion, and interaction between the membrane and polyelectrolytes doesno longer exist. In this way, cluster formation of lipids and associatedpermeabilization of the membrane can be circumvented.

Surprisingly, it has been found that liposomes including the substancesof the invention in the membrane thereof readily undergo fusion withother membranes, particularly cell membranes, below the isoelectricpoint of the substance. In general, this step requires the presence of alarger amount of PE in the membrane. As a result of its tendency offorming hexagonal phases, said PE assumes the function of a helperlipid. However, the inferior stability of such membranes isdisadvantageous, and gradual release of entrapped active substances isfrequently observed.

Advantageously, liposomes produced using the substances according to theinvention undergo effective fusion in the absence of helper lipids.Thus, when using the substances of the invention, it is possible toproduce liposomes which are capable of stably encapsulating an activesubstance, but undergo fusion with cell membranes under the conditionsof low pH values to release the active substance there.

This combination of two properties is an important precondition for theincorporation of cargo molecules in cells. In fusion of liposomes withcell envelopes or components, the aqueous volumes of both partnerscombine, with no opening of the membrane structures to the medium takingplace. As a result, uncontrolled influx or efflux of other substances isavoided.

In a preferred embodiment of the invention, the amount of sterolderivative is 50 mole-% at maximum. Compositions comprising at least 2mole-% of sterol derivatives, but 50 mole-% at maximum, are particularlyadvantageous. Compositions comprising at least 10 mole-% of sterolderivatives and 40 mole-% at maximum are also preferred. The productionof liposomes comprising the substances of the invention proceedsaccording to techniques well-known to those skilled in the art.

In another preferred embodiment of the invention, the liposomes comprisephosphatidyl choline, phosphatidyl ethanolamine, diacylglycerol,tetraether lipid, and/or PEG lipid. Cholesterols themselves areincapable of forming liposomes, and therefore, addition of further lipidis advantageous. This lipid can be any phospholipid. Obviously, thislipid can also be a ceramide or sphingolipid. It may be convenient tomodify the lipid with polyethylene glycol in the polar portion thereof.

In a preferred embodiment of the invention, the liposomes have anaverage size of between 50 and 1000 nm, preferably between 50 and 300nm, and more preferably between 60 and 130 nm.

Conveniently, active substances are enclosed in the liposomes, whichactive substances can be used e.g. in cancer therapy and in the therapyof severe infections. To this end, liposome dispersions can be injected,infused or implanted. Thereafter, they are distributed in the blood orlymph or release their active substance in a controlled fashion as adepot. The latter can be achieved by highly concentrated dispersions inthe form of gels. The liposomes can also be used for topical applicationon the skin. In particular, they may contribute to improved penetrationof various active substances into the skin or even passage through theskin and into the body. Furthermore, the liposomes can also be used ingene transfer. Due to its size and charge, genetic material is usuallyincapable of entering cells without an aid. For this purpose, suitablecarriers such as liposomes or lipid complexes are required which,together with the DNA, are to be taken up by the respective cells in anefficient and well-directed fashion. To this end, cell-inherenttransport mechanisms such as endocytosis are used. Obviously, theliposomes of the invention can also be used as model membranes. In theirprincipal structure, liposomes are highly similar to cell membranes.Therefore, they can be used as membrane models to quantify thepermeation rate of active substances through membranes or the membranebinding of active substances. More advantageously, the active substanceis a protein, a peptide, a DNA, RNA, an antisense nucleotide, and/or adecoy nucleotide.

In another preferred embodiment of the invention, at least 80% of theactive substance is entrapped in the liposomes. If necessary,non-incorporated cargo molecules adhering on the outside can be removedby simply increasing the pH value. This step is necessary in all thosecases where non-incorporated cargo molecules would give rise toaggregation of the liposomes. One advantageous fact when using thecomponents of the invention is that the entrapped active substances mustbe maintained under conditions allowing interaction with the lipid layeronly during the period of actual enclosure. Once the lipid layer remainsclosed in itself, it is possible to change to other conditions. Thereby,possible inactivation of active substances, particularly of proteins,can be minimized.

The invention also relates to methods of loading liposomes with activesubstances, using a binding pH value for encapsulation and a second pHvalue to remove unbound active substances.

Furthermore, the invention relates to a method of loading liposomes withactive substances, wherein the liposomes are made permeable at aspecific pH value and subsequently sealed. In particular, changes inpermeability preferably can be used in a well-directed fashion inloading liposomes. To this end, an active substance to be enclosed canbe added to a medium under conditions of high permeability, followed byadjusting conditions of low permeability. In this way, the activesubstance will remain inside the liposomes. Thereafter, non-entrappedactive substance can be removed, if necessary. Such changes inpermeability can be induced on liposomes or on liposomal nanocapsules.

The invention also relates to the use of the liposomes in diagnosticsand in release systems. Obviously, the liposomes can also be used in adetection system. In particular, the liposomes can be loaded with metalions whose fluorescence is enhanced by chelate formation, i.e., terbiumor europium ions, for example. Liposomes for such uses may of courseinclude components determining the specificity, i.e., antibodies,lectins, selectins, receptors, or hormones, or RNA aptamers. In aparticularly preferred embodiment of the use according to the invention,the presence of these metal ions is restricted to the volume of theliposomes so as to avoid non-specific signals from slowly released metalions adhering on the outside. It is also convenient to use the liposomesin the production of nanocapsules. The liposomes can be used withadvantage in the production of release systems in diagnostics. The usefor transport and/or release of active substances is also convenient.Advantageously, the liposomes can be used as depot formulation and/or ascirculative depot. The use of the liposomes as a vector to transfectcells in vivo, in vitro and/or ex vivo is also advantageous. Forexample, the liposomes can be used in intravenous and/or peritonealapplication.

The sterol derivatives and liposomes according to the invention involveseveral advantages. Surprisingly, it has been determined that thepermeability of the inventive liposomes depends on the pH value andthus, on the state of charge of the sterol derivative. When usingHist-Chol, for example, the liposomes comprising a phosphatidylethanolamine are made permeable at a pH value of from 5 to 6 in such away that entrapped active substances or markers will diffuse out withinminutes to hours. In other pH ranges, however, these membranesthemselves are stable, showing low initial permeability. Liposomes usingthe structures according to the invention are therefore particularlysuited to construct release systems wherein release of active substancesis to proceed in dependence on the pH value of the medium. Surprisingly,it has also been found that amounts of proteins or DNA above average canbe enclosed in liposomes including sterol derivatives in the membranesthereof. The efficiency of such incorporation depends on the pH value ofthe solution employed. Therefore, a process for efficient encapsulationof proteins or DNA in liposomes can be performed by initially adjustinga pH value that would result in good binding of the cargo molecules tothe lipid layer. With DNA as polyanion, low pH values of about 4 to 5are used. With proteins, a useful pH value will depend on theisoelectric point of the protein, which should be below the isoelectricpoint of the substance according to the invention. Encapsulation isparticularly effective when the pH value of the medium is selected so asto range between the isoelectric point of the protein and theisoelectric point of the sterol derivative. The protein then will have anegative and the lipid layer a positive charge.

Surprisingly, it has also been found that liposomes including e.g.Hist-Chol in the membranes thereof are capable of chelating metal ions.This property results in an increase of the positive charge of theliposome. This effect is observed to be particularly strong at neutralpH values, because the inherent charge of the compound is low in thiscase. Owing to their chelating properties, such liposomes can be used inbiochemical diagnostics and in pharmaceutical therapy.

One essential precondition for the use of liposomes for experimental ortherapeutic purposes is their compatibility with cells and tissues. Anumber of well-known compounds used to incorporate DNA or proteins incells (for example, the cationic lipid DOTAP) are cytotoxic.Surprisingly, it has been found that some of the compounds of theinvention exhibit reduced cytotoxicity. In particular, this applies tothat group of compounds wherein the amphoteric substance is an aminoacid or a peptide. These compounds therefore satisfy one of thepreconditions of a transfection system.

Another precondition for the construction of vectors to be used in geneor protein transport into cells is their compatibility with serum orblood. Due to their strong cationic charge, vectors known at presentform uncontrollable large aggregates, resulting in formation of thrombiin the organism. Their use in vivo is therefore practically impossibleand is restricted to in vitro or ex vivo applications. Surprisingly, ithas been found that liposomes constructed using the components of theinvention do not form any aggregates in serum or blood. In particular,these are liposomes having an isoelectric point below 7.5.

Another precondition for the construction of vectors to be used inprotein or gene transfer is their stability under physiologicalconditions. Upon application into the blood circulation, liposomes areattacked by components of the complement system and undergo rapid lysis.This reaction proceeds within minutes. As a result, pores are formed inthe membrane, which allow even large molecules such as proteins todiffuse out therethrough. At present, stabilization of liposomes withrespect to this mechanism is only possible by incorporating cholesterolin the lipid layer. While such liposomes are highly stable, they are nolonger able to interact with cells or readily release their activesubstance. Surprisingly, it has been found that liposomes constructedusing the components of the invention can be stable in serum or bloodfor several hours. Even under such conditions, the release of activesubstance is low. A liposomal vector for the transport of activesubstances must satisfy at least three preconditions: it must have lowtoxicity, entrap the active substance firmly and stably, and becompatible with serum or blood.

Advantageously, all of these three preconditions are satisfied byliposomes produced using selected substances according to the invention.The liposomes are therefore well suited for therapeutic uses. Otherproperties supporting such uses are good loadability with activesubstances and well-directed removal of these substances by changing thepH value or by permeabilization of the membrane. Liposomes producedusing the substances of the invention show low non-specific binding tocell surfaces. It is this low non-specific binding which is an essentialprecondition for achieving specific binding to target cells. Targetcontrol of the vehicles is obtained when providing the above-describedliposomes with additional ligands. As a result, the active substance canbe accumulated specifically in such cells or tissues which exhibit apathological condition.

One important use of the substances according to the invention istherefore in the construction of vectors for transfer of activesubstances in living organisms. The vectors are particularly suited forthe transport of therapeutic macromolecules such as proteins or DNAwhich themselves are incapable of penetrating the cell membrane orundergo rapid degradation in the bloodstream.

Without intending to be limiting, the invention will be explained inmore detail with reference to the following examples.

EXAMPLE 1 Synthesis of Cholesteryl-L-histidyl Succinate (Hist-Chol)

Step I, activation of cholesteryl hemisuccinate:Dicyclohexylcarbodiimide (11.3 mmol; 2.3 g) and 40 ml of THF are placedin a 100 ml flask and cooled to −10° C. Cholesterol hemisuccinate (10.3mmol; 5 g) and N-hydroxysuccinimide (11.3 mmol; 1.3 g) are added. Themixture is allowed to thaw to RT and stirred for 5 hours. Subsequently,the precipitate having formed (urea) is removed, the filtrate isconcentrated, and the residue is recrystallized from ethyl acetate.Colorless needles, m.p.: 145-146° C., 93%.

Step II, cholesteryl-L-histidyl succinate: the activated ester (5.1mmol; 3 g; step I) is placed in 40 ml of DMF. Sodium hydrogen carbonate(7.6 mmol; 0,6 g) and histidine (7.6 mmol; 1,2 g) are dissolved in 10 mlof water. This solution is added slowly and dropwise to the DMFsuspension. This is stirred for 20 hours and subsequently adjusted to pH4-5 using 2N HCl. The reaction mixture is then concentrated to dryness,and the product is extracted with hot ethanol. Following removal of theethanol, the product is obtained in pure form. Colorless solid, 78%.

EXAMPLE 2 Preparation of Amphoteric Liposomes

5 mg of Hist-Chol and 9.8 mg of POPC are dissolved in 4 ml ofchloroform/methanol (1:1 v/v) and dried completely in a rotaryevaporator. The lipid film is hydrated with 4.3 ml of a correspondingbuffer (10 mM Kac, 10 mM HEPES, 150 mM NaCl, pH 7.5) at a lipidconcentration of 5 mM using ultrasonic treatment for 5 minutes. Finally,the suspension is frozen and, following thawing, subjected to multipleextrusions (Avestine LiposoFast, polycarbonate filter, pore width 200nm). The profile of the zeta potential in mV at various pH values isillustrated in Table 2 below.

TABLE 2 pH value 0 mM NaCl 100 mM NaCl 4.0 +42 −20 5.0 +28 +2 6.0 −5 −67.0 −32 −15 8.0 −45 −25

EXAMPLE 3 Permeability

Lipid films having the composition DMPE/Hist-Chol 60:40 mole- % wereprepared in an analogous fashion as in Example 2 and hydrated using 100mM 6-carboxyfluoresceine (CF), 50 mM NaCl, HEPES 10 mM, pH 8, so as tomake a lipid concentration of 25 mM. Non-entrapped CF was removed by gelfiltration. For pH- and time-dependent permeability measurements, theliposomes were diluted to 0.2 mM in buffer of the respective pH, and thefluorescence was measured after 30 and 60 minutes, respectively.Thereafter, the temperature was raised to 37° C., and measurement waseffected at the same time intervals (30, 60 min). Subsequently, thetemperature was raised to 60° C. and again, measurement was effectedafter 30 and 60 minutes. The percent release of CF is summarized inTable 3 below. Permeabilization is clearly seen at pH 6.5, a value veryclose to the isoelectric point of Hist-Chol. The membranes aresurprisingly stable above and below this pH value.

TABLE 3 30 min 60 min 30 min 60 min 0 min 30 min 60 min (37° C.) (37°C.) (60° C.) (60° C.) pH 8.0 29% 29% 28% 30% 30% 36% 41% pH 7.0 32% 36%46% 39% 44% 52% 60% pH 6.5 53% 70% 81% 110%  132%  185%  180%  pH 6.029% 25% 26% 26% 27% 30% 33% pH 5.5 28% 26% 27% 26% 27% 30% 32% pH 5.039% 41% 43% 52% 61% 98% 115% 

EXAMPLE 4 Binding of DNA to Amphoteric Liposomes

For DNA binding, the liposomes comprised of POPC/Hist-Chol 60:40 (mole-%), prepared in Kac¹⁰HEP¹⁰NaCl¹⁰⁰, pH 8, according to Example 2, werediluted to 0.2 mM with the buffer to be investigated. DNA (herring DNA,1 mg/ml standard solution in water) was supplied in appropriate amounts.Subsequently, 0.2 mM liposomes (1 ml) were added. The mixture wasrapidly mixed and after 15 minutes, the particle size and zeta potentialwere determined by dynamic light scattering. With properly selectedamount of DNA, the particle size remains nearly unchanged.

At pH values of 7 and 8, where the POPC/Hist-Chol 60:40 liposomes have anegative charge, no change in size or zeta potential could be seen, thusexcluding binding of DNA. At pH 4 and 5, the liposomes have a strongpositive charge, and DNA binding results in charge exchange of theparticles. At pH 6, the liposomes have only weak charge, and a strongnegative charge upon addition of DNA. A considerably higher amount ofDNA must be added to obtain charge exchange. The zeta potentials and theassociated amounts of adsorbed DNA are summarized in Table 4 below:

TABLE 4 Zeta Adsorbed potential amount of DNA pH value DNA (mV)without/with (μg/mg lipid) 4 41.3 −37.7 15 5 19.6 −44.7 15 6 −4.2 −36.740 7 −36.8 −35 — 8 −52.2 −52.9 —

As can also be deduced from the binding characteristics, removal ofnon-entrapped DNA adhering on the outside can be achieved by increasingthe pH.

EXAMPLE 5 Stability in Human Serum

Liposomes of composition POPC/Hist-Chol 60:40 were prepared as a 5 mMsuspension in analogy to Example 2. The serum test was carried out as adilution series. Varying amounts of liposomes (50-250 μl) were incubatedwith an equal volume of human serum (250 μl) for 5 minutes at 37° C. (ineach case, the total volume was adjusted to 500 μl by addition of 150 mMNaCl). To measure the particle size by means of dynamic lightscattering, the mixture was then diluted 1:9 with buffer (KAc10 Hep10NaCl100; pH 8). The data are summarized in the Table below. The measuredparticle size approaches the serum value with increasing concentrationof the serum. Formation of large aggregates (>1 μm) was not observed(Table 5).

TABLE 5 Sample 250 μl of serum + x μl Particle size of liposomes (nm)Polydispersity 250 94 0.574 125 89 0.584  50 81 0.589 Serum only 700.613 Liposomes only 119 0.155

EXAMPLE 6 Pharmacokinetics in Blood

500 μl of liposomes comprised of POPC/Chol and POPC/Hist-Chol wereadministered to male Wistar rats by injection into the tail vein.

50 mM liposome suspensions were prepared by hydrating a lipid film ofthe respective formulation (addition of 0.03 mole-% of ¹⁴C-DPPC) with 2ml of a solution of 1 mg of ³H-inulin in HEPES 10 mM, NaCl 150 mM, pH 8.Following 3 freeze/thaw cycles, the suspensions were extruded severaltimes through a 400 nm membrane (LiposoFast, Avestin). Removal ofnon-entrapped ³H-inulin was effected by gel filtration over a G-75Sephadex column and subsequent concentrating over CENTRIPREP (Millipore)centrifugation units. 0.5 ml of liposome suspension was administered tofour test animals per formulation, and blood samples were taken after 5min, 15 min, 60 min, 3 hours, 12 hours, 24 hours. About 50 to 100 mg ofthe blood samples were dissolved in 1 ml of SOLVABLE tissue dissolver(PACKARD) at 50° C. for 1-3 hours and subsequently decolorized with0.1-0.5 ml of a 30% hydrogen peroxide solution. Thereafter, 10 ml ofscintillator was added, and the activity of ³H and ¹⁴C was measured. Nodirect toxic effects of the compounds could be detected.

Half-life of elimination from blood:

POPC/Chol >120 min POPC/Hist-Chol  >90 min

1. A sterol derivative according to formula (1):Amphoteric substance -Y- spacer -X- sterol  (1), wherein: saidamphoteric substance comprises a first portion having a cationic chargewith a pKa value between about 4 and about 8.5 and a second portion ofanionic charge with a pKa value between about 3 and about 7; said firstportion of said amphoteric substance is selected from the groupconsisting of piperazines, imidazoles, morpholines, purines, andpyrimidines; said second portion of said amphoteric substance comprisesa carboxyl group; said spacer is a linear or branched C₁₋₈ alkylcomprising 0-2 ethylenically unsaturated bonds; said linking group X isselected from the group consisting of —(C═O)—O—, —(C═O)—NH— and—NH—(C═O)—O—; said linking group Y is selected from the group consistingof —O—(C═O), —NH—(C═O), —(C═O)—O— and —(C═O)—NH—; said sterol isselected from the group consisting of cholesterol, sitosterol,campesterol, desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol,stigmasterol, 22-hydroxycholesterol, 25 -hydroxycholesterol, lanosterol,7-dehydrocholesterol, dihydrocholesterol, 19-hydroxycholesterol,5α-cholest-7-en-3β-ol, 7-hydroxycholesterol, epicholesterol, ergosterol,and dehydroergosterol; and said sterol derivative has an isoelectricpoint between about 4.5 and about 8.5.
 2. The sterol derivativeaccording to claim 1 wherein said amphoteric substance comprises 1-3portions of cationic charge and 1-3 portions of anionic charge.
 3. Thesterol derivative according to claim 1 wherein said sterol derivativehas an isoelectric point between about 5 and about
 7. 4. The sterolderivative according to claim 1 wherein said amphoteric substancecomprises a peptide of 1-10 amino acids, wherein said peptide comprisesas a charge carrier one or more amino acids selected from the groupconsisting of histidine, arginine, lysine, glutamic acid, and asparticacid.
 5. The sterol derivative according to claim 4 wherein the sum ofglutamic acid and aspartic acid amino acids of said peptide is greaterthan the sum of arginine, lysine, and histidine amino acids of saidpeptide.
 6. The sterol derivative according to claim 4 wherein saidpeptide comprises solely histidine as cationic amino acid, and whereinthe sum of glutamic acid amino acids and aspartic acid amino acids isgreater than or equal to the number of histidine amino acids.
 7. Aliposome comprising the sterol derivative of claim
 1. 8. The liposomeaccording to claim 7 wherein said liposome comprises less than about 50mole- % of sterol derivative.
 9. The liposome according to claim 8wherein said liposome comprises between about 2 mole- % and about 50mole- % of sterol derivative.
 10. The liposome according to claim 9wherein said liposome comprises between about 10 mole- % and about 40mole- % of sterol derivative.
 11. The liposome according to claim 7,wherein the liposome comprises one or more lipids selected from thegroup consisting of phosphatidyl choline, phosphatidyl ethanolamine,diacylglycerol, tetraether lipid, and PEG lipid.
 12. The liposomeaccording to claim 7 wherein said liposome has an average size ofbetween about 50 and about 1000 nm.
 13. The liposome according to claim12 wherein said liposome has an average size of between about 50 andabout 300 nm.
 14. The liposome according to claim 13 wherein saidliposome has an average size of between about 60 and about 130 nm. 15.The liposome according to claim 7 wherein said liposome furthercomprises an active substance.
 16. The liposome according to claim 15wherein said active substance is selected from the group consisting of aprotein, a peptide, a DNA, an RNA, an antisense nucleotide, a decoynucleotide, and a mixture thereof.
 17. The liposome according to claim15 wherein at least about 80% of said active substance is entrappedinside the liposome.
 18. A method of loading the liposome according toclaim 15 with said active substance, said method comprising:encapsulating said active substance in said liposome at a binding pHvalue; and removing unbound active substances at a second pH value. 19.A method of loading the liposome according to claim 8 with an activesubstance, said method comprising: permeabilizing said liposome bytreatment at a pH value sufficient to enable loading of said activesubstance, and sealing said liposome.
 20. A method for the transport andrelease of an active substance in a subject, said method comprisingadministering to said subject the liposome of claim
 15. 21. The methodof claim 20, wherein said administration is intravenous or peritoneal.22. A depot formulation or circulative depot comprising the liposome ofclaim
 7. 23. A nanocapsule prepared from the liposome of claim
 7. 24. Avector for transfecting cells in vivo, in vitro or at vivo, said vectorcomprising the liposome of claim 7 and a nucleic acid.